熱測定
Online ISSN : 1884-1899
Print ISSN : 0386-2615
ISSN-L : 0386-2615
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47 巻, 1 号
選択された号の論文の7件中1～7を表示しています
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特集－バイオカロリメトリー－
  • Isothermal Titration Calorimetry を用いた酵素に対する金属イオンの相互作用解析
    判谷 吉嗣, 千賀 明香音, 河合 富佐子
    2020 年 47 巻 1 号 p. 2-7
    発行日: 2020/01/25
    公開日: 2022/11/20
    DOIhttps://doi.org/10.11311/jscta.47.1_2
    ジャーナル フリー
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    Metal ions play essential roles in protein structures, function, and stability. Practically, over one third of proteins from Protein Data Bank (PDB) contain metal ions, and approximately 40 % of enzymes for which three-dimensional structures are known required metal ions for their activities. Metal ions play roles as cofactors for enzymes and regulate functions of enzymes by binding to the enzymes, except as cofactors. Therefore, knowledge of metal-enzyme interactions is essential for elucidating the function and reaction mechanism of enzymes. Metal-binding has been identified by such experimental approaches as isothermal titration calorimetry (ITC), circular dichroism, differential scanning calorimetry, absorbance spectroscopy, and X-ray. Among them, ITC is particularly a fundamentally essential technique to study metal-enzyme interactions, because it can reliably assess the thermodynamic underpinnings of binding events, and it is not restricted by the photophysical properties of a metal. In this review, we highlight the analysis of enzyme-metal interactions based on ITC about two enzymes, carbonic anhydrase II (CAII) and cutinase-like enzyme (Cut190), in which metal ions are playing different roles as a cofactor and activating/stabilizing factors, respectively. We evaluate and elucidate analytical results and propose the additional plans, which complement ITC in case metal binding is too weak for reliable assessment.
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  • タンパク質凝集系の熱量測定
    神山 匡
    2020 年 47 巻 1 号 p. 8-13
    発行日: 2020/01/25
    公開日: 2022/11/20
    DOIhttps://doi.org/10.11311/jscta.47.1_8
    ジャーナル フリー
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    In order to realize the mechanism of aggregate formation of proteins in solution, the thermodynamic properties and state diagram of the thermally induced aggregation of protein at various molar fractions of 1,4-dioxane were determined by DSC, circular dichroism (CD), dynamic light scattering (DLS), and scanning electron microscopy (SEM). In this solution, the transition temperatures of thermal denaturation and aggregation differ significantly, so each transition enthalpy can be easily determined. The transition temperature and enthalpy of protein were significantly dependent on the molar fraction of 1,4-dioxane, incubation time, and scan rate. In this paper, the DSC measurements for protein aggregation are introduced focusing on the recently revealed results for β-lactoglobulin (LG). The secondary structure of LG influenced greatly with dioxane concentration, temperature, and incubation time, resulting the aggregated structure became spherical or amorphous. The enthalpy changes for the thermal aggregation were large negative (exothermic) which may reflect to the aggregation from a supersaturated state of the protein and the interaction between protein molecules.
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  • 分子間相互作用解析プログラム“SEDPHAT”による等温滴定型熱量データ解析
    丸野 孝浩, 内山 進
    2020 年 47 巻 1 号 p. 14-18
    発行日: 2020/01/25
    公開日: 2022/11/20
    DOIhttps://doi.org/10.11311/jscta.47.1_14
    ジャーナル フリー
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    Isothermal titration calorimetry (ITC) directly provides the detailed thermodynamic characterization (Gibbs free energy ΔG, enthalpy change ΔH, entropy change ΔS, and the stoichiometry n) in solution. Since the release of commercially available ITC in the early 1990’s, the number of studies that have adopted this method has increased. In particular, ITC instruments with small sample volumes have the adequate sensitivity to measure the heat change upon interaction and have accelerated the application of this method in various fields, including biochemistry, medicinal chemistry, structural biology, and material sciences. In this paper, using program SEDPHAT which has attractive features for non-linear fitting of the binding isotherm, as representative cases of SEDPHAT analysis, we describe the ITC data analyses for A+B ‹−› AB and A+B+B ‹−› ABB systems and the global fittings.
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  • 断片抗体アフィニティー精製用リガンド・プロテインGの機能
    吉田 慎一
    2020 年 47 巻 1 号 p. 19-23
    発行日: 2020/01/25
    公開日: 2022/11/20
    DOIhttps://doi.org/10.11311/jscta.47.1_19
    ジャーナル フリー
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    Streptococcal protein G (SpG) is a bacterial surface protein, binding mainly to the Fc (high affinity) and Fab (low affinity) regions of immunoglobulins. The SpG-immobilized affinity chromatography resin is useful for the purification of antibodies. Improvement of the affinity of SpG to Fab enhances its application to the purification of Fab-based antibody fragments. We found four important mutations of SpG, improving its affinity for Fab through affinity maturation using a ribosomal display system. In this article, we overview the calorimetric analyses of these mutations on the function and stability of SpG as an affinity ligand. Isothermal titration calorimetry analysis elucidated the quantitative enthalpic/entropic contributions of these individual mutations on the interaction of SpG with Fab. Differential scanning calorimetry analysis revealed the destabilizing effects of these mutations on the thermodynamic stability of SpG. These calorimetric analyses may contribute to the current understanding of the mechanism of action of such mutations and further refinement of affinity ligands through protein engineering. Additionally, we discuss key features of our new SpGimmobilized affinity chromatography resin such as binding ability and purity profile of target molecules.
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